The present invention relates generally to an apparatus such as a spectrophotometer for automatically and simultaneously determining the relative concentration of two samples or solutions. More particularly the present invention may be used for automatically determining the amount of a hemoglobin species in a blood sample relative to the total hemoglobin of the blood sample. The present invention, of course, has a broader applicability than merely hemoglobin ratios. In essence, when measuring the concentrations of two or more solutions for the purpose of an ultimate comparison, it is important that the spectrophotometer not drift or deviate or vary between readings. The present invention overcomes this problem by providing simultaneous measuring of the concentration.
To aid in a complete understanding of the benefit of the present invention, a theoretical explanation of the blood chemistry evaluation will be given. It is known, for example, to prepare blood samples for hemoglobin analyses by a procedure called column chromatography. Column chromatography is used in clinical laboratories to determine levels of various species of hemoglobin in the blood. One such hemoglobin species is Hemoglobin A.sub.1c (HbA.sub.1c). It has been shown by various investigators that the level of HbA.sub.1c circulating in the blood is an indicator of the status of glucose metabolism in individuals. Determining the percent of the HbA.sub.1c component relative to total hemoglobin aids in the diagnosis and treatment of diabetes (see, for example, R. J. Koenig, C. M. Peterson, et al, New England Journal of Medicine, Vol. 295, pp 417-419, Aug. 19, 1976). Methods have been developed for the column chromatographic separation of HbA.sub.1a, HbA.sub.1b and HbA.sub.1c as a group from the rest of the hemoglobins using cation exchange chromatography, and the major component of this HbA.sub.1 group is HbA.sub.1c. In this technique, the cation exchange may include use of either a cellulosic or non-cellulosic or non-cellulosic cation resin. For example, cellulosic resins were used by Huisman, et al, Clin. Chim. Act., Vol. 5, pp. 103-123, 1960. Additionally, use of cellulose cation exchange resins for the determination of HbA.sub.1 is the subject of U.S. Pat. Nos. 4,142,855, 4,142,856 and 4,142,857. Non-cellulosic cation exchange columns are described by Trivelli, et al, New England Journal of Medicine, Vol. 84, pp. 353-357, February 1971, and by M. D. Clegg and W. A. Schroeder, Journal of the American Chemical Society, Vol. 83, pp. 1472-1478, 1961, and are the subject of U.S. Pat. No. 4,142,858. These procedures all require separation of two blood fractions using an ion-exchange column, followed by separate measurements and calculations to arrive at a numerical value of HbA.sub.1.
Still another hemoglobin species is Hemoglobin A.sub.2 (HbA.sub.2). Determining the amount of HbA.sub.2 present in blood provides an aid to the physician in diagnosing disorders of the red blood cells, especially the genetic disorder B-Thalassemia. HbA.sub.2, like HbA.sub.1, may be determined by column chromatographic techniques using ion-exchange chromatography (see for example, E. C. Abraham, Hemoglobin, Vol. 1, pp. 27-44 1976). As in the HbA.sub.1c procedure, the HbA.sub.2 procedure requires many manipulations and measurements following the step of chromatographic separation.
In order to understand the nature of the calculations and manipulations which must be made after the column chromatographic separation, it should be understood that when measuring the concentration of a substance, such as hemoglobin in solution, the concentration of the substance measured photometrically usually follows Beer's Law where the negative logarithm of the transmittance of light through the sample varies as a function of the concentration. Specifically, the formula is log (I/I.sub.o)=-kC where the ratio (I/I.sub.o) is referred to as the transmittance or percent of light which is not absorbed by the sample. Thus, in the formula, the transmittance I/I.sub.o is the ratio of the intensity of a beam of light passing through the solution divided by the intensity of the same beam of light passing through a solution containing a zero concentration of the substance to be measured. A solution containing a zero concentration of the substance to be measured is referred to as a "blank" solution.
Thus it may be appreciated that when the relative concentration of hemoglobins as between two samples is to be measured and calculated, the value of the transmittance, I/I.sub.o, must be determined for each sample.
For example, to determine the amount of hemoglobin HbA.sub.2 as a percent of the total hemoglobins in the blood, two samples must be prepared such as by column chromatography, the first sample containing only HbA.sub.2 and the second sample containing the remaining hemoglobins. Each sample is then separately placed in a spectrophotometer. For the first sample, the values I and I.sub.o are determined. Then the values I and I.sub.o are determined for the second sample. Then the percent of hemoglobin HbA.sub.2 is calculated according to the formula ##EQU1##
According to the prior art, each of the measurements required by the above formula were separately determined and this created inherent variations because of changes in the optical system such as those occasioned by drift of the spectrophotometer as well as degradation of the optical system. We have discovered that the problems of the prior art technique may be eliminated by the present invention which provides two channels to simultaneously measure the transmittance of the two samples using a single light source.
Another problem with prior art techniques was that even when the transmittance of a single sample was being determined there could be degradation of the optical system between the time of calibration of the system and the time at which the transmittance of light through a particular sample was being measured. The co-pending application referred to above addresses itself to an automatic blanking circuit for effectively self-calibrating the spectrophotometer automatically and the features of automatic blanking are preferably included in the method and apparatus of the present invention.
Thus the present invention provides an improved method and apparatus for determining the relative concentration of two samples by providing for the simultaneous measurement of the concentration of each sample using a single source of light.